The mammalian target of rapamycin, mTOR, is a key element in a signaling pathway that controls mRNA translation and cell proliferation. mTOR is a member of a family of protein Ser/Thr kinases that have catalytic domains homologous to that of the catalytic subunit of phosphatidylinositol 3-OH kinase. How the protein kinase activity of mTOR is controlled is not known, and the downstream effectors and targets in the mTOR pathway have not been fully defined. In Aim 1 of this proposal, the role of phosphorylation in controlling mTOR will be investigated. Insulin increases the protein kinase activity of mTOR and promotes the phosphorylation of one or more sites in a putative regulatory domain near the COOH terminus of mTOR. Binding of an antibody to this region also increases the kinase activity. We will attempt to determine whether phosphorylation of this domain directly activates mTOR or recruits an activating binding protein. The hypothesis that changes in the phosphorylation of sites in this domain are required for effects of insulin, amino acids or cAMP on cellular events controlled by the mTOR pathway will be tested. In Saccharomyces cerevisiae the effects of Tor1p and Tor2p, the yeast counterparts of mTOR, appear to be mediated by proteins homologous to protein phosphatase 2A (PP2A) and alpha4 protein. In Aim 2 the potential role of alpha4 as an effector of mTOR will be investigated. The hypothesis that mTOR- dependent phosphorylation of alpha4 decreases PP2A activity by promoting formation of an alpha4-PP2A complex will be tested. Aim 3 is to identify new downstream targets of the mTOR pathway. Chromatographic methods followed by either one- or two- dimensional electrophoresis will be used to resolve proteins whose phosphorylation is changed in response to rapamycin, a potent inhibitor of mTOR function. A newly developed method involving mixed peptide sequencing and data base searching will be used to identify the candidates. We hope that finding new targets will provide insight into the mechanisms involved in mTOR signaling and suggest new directions for future research.